Abstract
Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.
Highlights
Translin-Associated Factor X (TRAX) protein was initially identified in a yeast two hybrid screen as a protein that interacts with translin, a ssDNA and RNA binding protein [1,2,3]
The nuclease activity of translin-TRAX heteromeric complex has been in recent focus due to its role in siRNA processing [16,29]
Binding of DNA to the translin-TRAX complex has been thought to be mediated through translin, and TRAX was not expected to bind the nucleic acid substrates [21,22,29]
Summary
TRAX protein was initially identified in a yeast two hybrid screen as a protein that interacts with translin, a ssDNA and RNA binding protein [1,2,3]. Translin and TRAX proteins share homology and have been evolutionarily conserved in eukaryotes [4,5,6] Evidence that these endogenous proteins interact in vivo has been provided by immuno- precipitation studies [7,8]. As the translin-TRAX complex binds to both ssDNA and RNA, it has been implicated in a variety of cellular functions. It was thought that its ability to bind to ssDNA indicated its involvement in DNA repair [3,13] This view has been supported by the observation that DNA damaging agents induce TRAX’s interaction with the nuclear C1D as a part of a cellular DNA repair response [14]. TRAX has been found to suppress proliferation of PC12 cells in the presence of p53 blockade [17]
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